KR100936645B1 - Raman microscope - Google Patents
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- KR100936645B1 KR100936645B1 KR1020080041031A KR20080041031A KR100936645B1 KR 100936645 B1 KR100936645 B1 KR 100936645B1 KR 1020080041031 A KR1020080041031 A KR 1020080041031A KR 20080041031 A KR20080041031 A KR 20080041031A KR 100936645 B1 KR100936645 B1 KR 100936645B1
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- G02B21/0088—Inverse microscopes
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Abstract
The present invention relates to a Raman microscope, (i) a variable aperture type image selection mechanism (AP) for selectively passing only a desired portion of an inverted image of a sample obtained by the objective lens (L1), (ii) the image selection mechanism A short wavelength interfering visible light is removed from the wide magnification lens L2 for expanding the width of each of the Raman scattered light and the interfering visible light passing through the AP, and the Raman scattering light and the interfering visible light passing through the widening lens L2. The main light includes a long wavelength optical filter FL and a condenser lens L3 which induces a focal length difference between the Raman scattered light and the interference visible light to remove the interference visible light.
In addition, the present invention is further characterized by further comprising a visible light blocking plate BL positioned on the focus of the visible light passing through the condensing lens L3.
The present invention can obtain an excellent noise-to-signal ratio (S / N) spectrum by using a very small sample without being influenced by the use environment illumination light, and can reduce the limit size of the measurable sample to about 1 to 10 μm.
Raman microscope, S / N, image selection, visible light, wide magnification lens, optical filter, visible light blocking film.
Description
The present invention relates to a Raman microscope, and more particularly, to minimize visible light interference from an external lighting environment, so that an excellent noise-contrast signal when measuring a small amount of sample under normal laboratory lighting without using separate dark rooms or dark boxes is used. The present invention relates to a Raman microscope capable of obtaining a spectrum of a ratio of signal to noise (hereinafter referred to as "S / N").
Raman spectroscopy is a device for qualitative and quantitative analysis of materials using Raman scattering.The principle of measurement is that when monochromatic light such as a laser beam having a frequency of V0 is irradiated to a molecular bond vibrating at the frequency of V1, Most of the light is scattered as it is without changing the frequency, but some give V1 corresponding to the binding energy of the molecule to the molecular bond (V0-V1) or receive energy from the molecular bond (V0 + V1), and scattering occurs and the wavelength It becomes longer or the wavelength becomes shorter. This change in wavelength is called Raman scattering, and the change in wavelength corresponds to the infrared region, and every material has its own change value. Therefore, qualitative and quantitative analysis of the substance is possible from this change of wavelength such as human fingerprint.
The Raman spectrometer measures qualitatively and quantitatively this change value.
In quantum mechanics, according to Boltzmann distribution, the intensity of the stock-line is much higher than that of the antistock-line, so Raman spectroscopy, except in special cases, -line) for analysis.
At this time, since the change value of the frequency V1 shows different values depending on the substance, it becomes possible to perform qualitative and quantitative analysis of the unknown sample by measuring the degree of change and the intensity thereof.
Therefore, Raman scattering analysis is less sensitive than other spectroscopic methods, but if the amount of sample is sufficient, it is one of the popular methods because of many advantages such as convenience of sample preparation, rapid analysis, and selection of various media. In this case, it can be measured in a glass vial or with the external light blocked in the sample compartment or probe sampler box of the instrument. There is no.
However, when the sample size becomes smaller, it is a completely different situation. Especially, when the sample size becomes small enough to be seen only under the microscope field of view, it is difficult to measure by the general Raman spectroscopy method. There is no choice but to. The most common method for measuring a Raman of a micro sample is to use an optical microscope as a Raman microscope by connecting a Raman adapter to a general optical microscope as shown in FIG. 2.
The size relationship between the objective lens and the image of the convex microscope is as follows.
When a is larger than f and an image S1 is located at the front of the convex lens having a focal length of f, and the a is larger than f, the resulting image S2 is formed by the distance b obtained by the following equation (I), The size is according to the following formula (II).
1 / a + 1 / b = 1 / f (Ⅰ)
S2 / S1 = b / a (II)
However, the above case is a case of monochromatic light, and in the case of multicolor light such as white light, the refractive index is changed according to the wavelength of light, and thus the distance at which an image is formed varies depending on the wavelength. When light passes through a dense material, such as a glass lens, in air, the shorter the wavelength, the greater the refractive index and the shorter the location of the image. This is called chromatic aberration and becomes larger when using a single lens.
Therefore, if the image of the short wavelength light is S2 ', the distance is b', the image of the long wavelength light is S2 "and the distance b" is S2 '<S2 ", b' <b".
The conventional Raman microscope is used as a Raman microscope by attaching a Raman adapter to a general optical microscope as shown in Figure 2, the operation principle is as follows.
2 is a schematic view showing the basic structure of a conventional Raman microscope.
Sample S is located at the focal length of objective lens L4, and the light from the sample becomes parallel light.
This parallel light forms an inverted image at the focal point F by the condensing lens L5, and is positioned directly within the focal length of the alternative lens L6, so that the enlarged inverted virtual image can be viewed.
Therefore, even if the distance between the two lenses is changed by installing the Raman adapter R between the condenser lens L5 and the objective lens L4, operation as an optical microscope is not affected.
After installing the Raman adapter R in this way, a monochromatic laser light source is incident on the side of the microscope, and the sample is focused on the sample at the focal position with the objective lens L4 for irradiation. After that, the Raman scattered light generated from the sample is sent through the same path to the spectrometer for measurement. At this time, in the sample S, not only the Raman scattered light (L) by the irradiated laser light but also the illumination light (v) of the environment surrounding the device is raised with the sample. This is also sent to the Raman spectroscopy, which acts as a disturbing light in the Raman spectrum. At this time, if the size of the sample is smaller than the beam diameter of the laser light source to be irradiated, the S / N of the sample itself is worse, but the influence of external light is greater, especially when the size of the sample is extremely small, such as 1 to 10 microns. It is almost impossible to obtain the spectrum of the sample.
Therefore, in order to exclude the influence of the ambient light, the measurement should be carried out in a dark room environment in which the ambient light is eliminated or the instrument itself should be placed in a dark box so that the ambient light is not irradiated to the sample.
The present invention solves the interference problem of the external illumination light, which is the biggest problem of the conventional Raman microscope, while lowering the measurement limit size of the sample to 1 to 10 microns in a general laboratory environment in which normal illumination is performed without using a dark room or a dark box. We present a Raman microscope with an excellent signal to noise ratio (S / N) and ease of use.
Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.
First, the Raman microscope according to the present invention, as shown in Figure 1, using the laser light source as one selected from the objective lens (L1) to obtain an inverted real image of the Raman spectrograph and a device for visually confirming the sample state In the Raman microscope to be sent to the apparatus of (i) a variable aperture type image selection mechanism (AP) for selectively passing only the desired portion of the inverted real image of the sample obtained by the objective lens (L1), (ii) the image selection A wide wavelength lens L2 which enlarges the width of each of the Raman scattered light and the interference visible light passing through the mechanism AP, and (i) interfering visible light having a short wavelength among the Raman scattered light and the interference visible light passing through the wide magnification lens L2. And a condensing lens L3 which removes the interference visible light by causing a long wavelength optical filter FL to remove and (i) a Raman scattered light and a focal length difference between the interference visible light. Shall be.
1 is a schematic view showing the basic structure of a Raman microscope according to the present invention.
In general, as described above, the Raman microscope uses the laser light as a light source to obtain an inverted image of the sample, and then sends the image to the Raman spectrometer to measure the S / N spectrum or to visually check the sample state.
The present invention also uses a laser light (L) as a light source as shown in FIG. 1 to obtain an inverted real image of the sample with the objective lens (L1) and then one of the apparatus selected from the visual inspection of the Raman spectroscopy and the sample state Send it to
As shown in FIG. 1, the present invention provides a variable aperture type image selection mechanism AP for selectively passing only a desired portion of an inverted image of a sample obtained by the objective lens L1, and (ii) the image selection. A wide-wavelength magnifying lens L2 that enlarges the width of each of the Raman scattered light and the interference visible light that has passed through the mechanism AP, and (i) interfering visible light having a short wavelength among the Raman scattered light and the interference visible light that have passed through the widening lens L2. And a condensing lens L3 that removes the interference visible light by causing a long wavelength optical filter FL to remove and (i) a Raman scattered light and a focal length difference between the interference visible light.
The image selection mechanism AP is a variable aperture type that can change the size continuously or stepwise, and has a circular or polygonal shape.
In addition, the Raman microscope of the present invention may further include a visible light blocking plate BL positioned on the focus Fi2 of the visible light passing through the condenser lens L3.
The visible light blocking plate BL is in the form of a circle or a polygon.
The size of the visible light blocking plate BL is preferably a focal size of visible light passing through the condensing lens L3.
Hereinafter, an example of a Raman microscope according to the present invention will be described in more detail with reference to FIG. 1.
The solid line L shown in FIG. 1 represents the laser irradiation light and the Raman scattering signal light, and the dotted line V represents the interference light in the visible light band coming from the illumination of the environment around the device.
First, the monochromatic laser light L from the laser light source is transmitted by a beam splitter (BS), about half of which is reflected, and about half of which is reflected and bent at 90 degrees to fall perpendicular to the objective lens L1. This light is condensed by the objective lens L1 and irradiated to the sample S. In this case, if the size of the sample is larger than the size of the laser light, there is no difficulty, but if the size of the sample is smaller than 10 microns or less, as shown in FIG. 3, the external illumination visible light may come up the same path as the Raman signal. .
3 is a schematic diagram showing a process in which external illumination light enters from an objective lens.
In more detail, the externally visible visible light can enter only from the side of the objective lens L1. The maximum incident angle of the light and the reflection angle θ are the incident angle or the reflection angle which is not hidden by the objective lens L1. Therefore, it is difficult for the visible light to rise toward the objective lens, but if the reflected light is diffusely reflected at the surface of the sample, it will travel in the same path as the Raman scattering signal light and affect it. Of course, the light is very weak, but the Raman signal light itself is very weak, which is enough to adversely affect it. Therefore, in order to obtain a good Raman spectrum, it is necessary to remove the visible light such as Raman scattered light.
The process of removing the external illumination visible light is as follows.
The ambient visible light and the Raman scattering signal light starting from the sample S form the visible light inverted phase Si and the Raman scattered light inverted phase Sr at the positions of the visible light primary focus Fi1 and the Raman scattered light primary focus Fr1 by the objective lens L1. In this case, a stock-line having a longer wavelength is used among the Raman scattered signal lights. In this case, the Raman scattered light has a longer wavelength than the visible visible light, and thus forms an upper phase due to chromatic aberration.
Next, only the portion of the inverted image that is desired to be measured is cut out with an image selector AP of a variable aperture type to remove visible light or unnecessary portions raised from the periphery of the sample. Nevertheless, the peripheral illumination visible light scattered from the sample surface is only on the same axis as the Raman scattering signal light and differs only in focal distance, and thus cannot be removed by the variable aperture type image selection device AP.
The Raman scattering signal light and the interfering visible light passing through the image selection device AP are widened by the wider lens L2.
That is, the distance from the visible light primary focus Fi1 and the Raman scattered light primary focus Fr1 to the wide magnification lens L2 is shorter than the focal length of the wide magnification lens L2 so that the light continues to spread. The visible light inverted real image Si is farther from the wider magnification lens L2 and has a larger refractive index than the Raman scattered light inverted real image Sr, so that visible light spreads less than the Raman scattered light. In addition to the purpose of separating the focus, the wider width also increases the efficiency of the long pass filter FL. That is, the wider lens is enlarged by the wider lens L2 and the angle of incidence is adjusted to effectively pass a large area of the long pass filter FL while cutting light in a region shorter than the wavelength of the used light source laser beam and passing only a long wavelength region. At this time, the visible light is also removed.
After the light passes through the long pass filter FL, a small amount of visible light is removed by the re-condensing process of the condenser lens L3. That is, the Raman scattering signal light having a long wavelength and the ambient visible light having a short wavelength pass through the condensing lens L3, and the focus is separated when the light is collected again. The visible light of the short wavelength is first collected and spread, and the Raman scattering signal light is focused upward. By using the difference in focal length, the Raman scattering signal light has a high degree of integration and a good signal can be obtained by matching the inlet of the optical sensor N1 connected with the detector to the focal position of the Raman scattering signal light, while the visible light has already been enlarged beyond the focus. Its low density and improper angle of incidence of light make it possible to remove significant levels.
If visible light still remains to affect the Raman scattering signal light, it is preferable to provide a visible light blocking plate BL on the focus Fi2 of visible light passing through the condenser lens L3 to increase the visible light removal rate.
The size of the visible light blocking plate BL is preferably a focal size Fi2 of visible light passing through the condenser lens L3, and the shape is circular or polygonal.
The present invention can obtain an excellent noise-to-signal ratio (S / N) spectrum by using a very small sample without being influenced by the use environment illumination light, and can reduce the limit size of the measurable sample to about 1 to 10 μm.
Hereinafter, the present invention will be described in more detail with reference to the following examples.
However, the present invention is not limited only to the following examples.
Example One
In the Raman microscope of the present invention as shown in FIG. 1, a polyphenylenesulfide having a size of about 2 microns is placed on a slide glass (SG) as shown in FIG. 4, and a sample (S) covered with a cover glass (CG) is generally used. Measurements in the laboratory under the lighting environment resulted in a Raman spectrum as shown in FIG. 5, which is in full agreement with the standard spectra of bulk samples of the same material and shows no influence of room illumination light.
Example 2
The Raman microscope of the present invention as shown in FIG. 1 measured a piece of acrylic having a size of about 5 microns placed on a glass plate in a general laboratory environment under illumination, and as a result, the Raman spectrum of acrylic without the influence of illumination light as shown in FIG. 6 was obtained. .
Example 3
The Raman microscope of the present invention as shown in FIG. 1 measured ammonium sulfate particles having a size of about 10 microns placed on a glass plate in a general laboratory environment to obtain a Raman spectrum without the influence of illumination light as shown in FIG. 7.
1 is a schematic view showing the basic structure of a Raman microscope according to the present invention.
2 is a schematic view showing the basic structure of a conventional Raman microscope.
3 is a schematic diagram showing a process in which external illumination light enters from an objective lens.
Figure 4 is a schematic diagram of a fine sample on a slide glass covered with a cover glass which is a sample of the present invention Example 1.
5 is a Raman spectrum of the polyphenylene sulfide measured in Example 1 of the present invention.
6 is a Raman spectrum of the acrylic resin measured in Example 2 of the present invention.
7 is a Raman spectrum of an ammonium sulfate sample measured in Example 3 of the present invention.
* Explanation of symbols on the main parts of the drawings.
S: sample f0: objective lens focus L1, L4: objective lens
f1: focus of the laser light passing through the objective lens
L: Laser light v: Visible light
BS: Beam splitter Fi1: Visible light primary focus
Fr1: Raman scattered light primary focus Si: visible light inverted image
Sr: Raman Scattered Light Inverted Reality AP: Image Selector (Variable Iris)
L2: Wide magnifying lens FL: Long wavelength filter
L3, L5: condenser lens Fi2: visible light secondary focus Fr2: Raman scattered light secondary focus
BL: Visible light blocker
L6: Alternative lens N: Raman spectroscopy detector
N1: Optical fiber sensor of Raman spectroscopy detector F: Focus of alternative lens
R: Raman adapter θ: Incident and reflection angles of visible light
SG: Slide Glass CG: Cover Glass
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KR1020080041031A KR100936645B1 (en) | 2008-05-01 | 2008-05-01 | Raman microscope |
PCT/KR2008/002592 WO2009133980A1 (en) | 2008-05-01 | 2008-05-08 | Raman microscope |
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KR1020080041031A KR100936645B1 (en) | 2008-05-01 | 2008-05-01 | Raman microscope |
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WO2015105457A1 (en) | 2014-01-13 | 2015-07-16 | Nanyang Technological University | Device for determining a condition of an organ and method of operating the same |
CN117783088B (en) * | 2024-02-23 | 2024-05-14 | 广州贝拓科学技术有限公司 | Control model training method, device and equipment of laser micro-Raman spectrometer |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS6117032A (en) | 1984-07-03 | 1986-01-25 | Nec Corp | Microscope-laser raman spectrometer |
KR960014969B1 (en) * | 1991-05-14 | 1996-10-23 | 로무 가부시기가이샤 | Microscope |
JPH08327550A (en) * | 1995-06-02 | 1996-12-13 | Tokai Carbon Co Ltd | Raman spectrometer |
US6909105B1 (en) | 1999-03-02 | 2005-06-21 | Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. | Method and device for representing an object |
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DE19630956A1 (en) * | 1996-07-31 | 1998-02-05 | Basf Ag | Method and device for Raman correlation spectroscopy |
US7039452B2 (en) * | 2002-12-19 | 2006-05-02 | The University Of Utah Research Foundation | Method and apparatus for Raman imaging of macular pigments |
EP1651109A1 (en) * | 2003-07-31 | 2006-05-03 | Koninklijke Philips Electronics N.V. | Method and apparatus for determining a property of a fluid which flows through a biological tubular structure with variable numerical aperture |
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- 2008-05-01 KR KR1020080041031A patent/KR100936645B1/en active IP Right Grant
- 2008-05-08 WO PCT/KR2008/002592 patent/WO2009133980A1/en active Application Filing
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
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JPS6117032A (en) | 1984-07-03 | 1986-01-25 | Nec Corp | Microscope-laser raman spectrometer |
KR960014969B1 (en) * | 1991-05-14 | 1996-10-23 | 로무 가부시기가이샤 | Microscope |
JPH08327550A (en) * | 1995-06-02 | 1996-12-13 | Tokai Carbon Co Ltd | Raman spectrometer |
US6909105B1 (en) | 1999-03-02 | 2005-06-21 | Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. | Method and device for representing an object |
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